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MARINE ECOLOGY PROGRESS SERIES Published December 31 Mar Ecol Proy Ser

Symbiosis between methane-oxidizing and a deep- carnivorous cladorhizid

Jean ~acelet'v*,Aline ~iala-~edioni~,C. R. is her^,Nicole Boury-Esnaultl

'Centre d'oceanologie de Marseille, Universite de la Mediterranee - UMR CNRS no. 6540, Station Marine d'Endoume, F-13007 Marseille, France 20bservatoireOceanoloyique. Laboratoire Arayo, F-66650 Banyuls-sur-Mer, France 3Department of , Pennsylvania State University, University Park, Pennsylvania 16802. USA

ABSTRACT Dense bush-l~keclumps of several hundred ~nd~vldualsof a new specles of (Demosponglae, Poeciloscler~da)were observed near methane sources in mud volcanoes 4718 to 4943 m dcep In the Barbddos Trench The sponge contalns 2 maln mo~pholog~caltypes of extra- cellular symbiotic bacteria small rod-shaped cells and larger cocco~dcells with stacked membranes Stdble carbon ~sotopevalues, the presence of methanol dehydrogenase and ultrastructural observa- tlons all lndlcate that at least some of the syrnblonts are methdnotrophic Ultrastructural ev~denceof ~nlracell~~lard~gest~on of thp syn~b~ontsand the stable C and X values suggest that thc sponge obtalns a slgnlflcdnt portion of its nutr~tlonfrom the symbionts Ultrdstructure of the sponqe embryo suggests d~recttransmission throuqh qc.neratlons In brooded embryos The sponge also ma~ntrilnsa carnivorous feed~nghdblt on tin\ swlmmlng prey, as do other cladorhlzlds

KEYL\/ORDS. Methanotrophy POI-~fera. Deep-sea . . Cold-seep communities

INTRODUCTION although they are sometimes abundant on extinct chimneys (Boury-Esnault & De Vos 1988) or in vent Associations between several phyla of marine inver- peripheral areas. The lack of in most tebrates and chemoautotrophic -oxidizing and/or chemosynthetic communities i.s rather surprising as methanotrophic bacteria are frequently found in they are probably the marine in which marine communities surrounding hydrothermal vents, symbiosis with the widest variety of microsymbionts, cold seeps, and other reducing envi.ronments (Chil- and heterotrophic bacteria occurs (Vacelet & dress et al. 1986, Fiala-Medioni & Felbeck 1990, Donadey 1977, Wilkinson 1987, 1992). All sponge spe- Cavanaugh 1993, Nelson & Fisher 1995). These associ- cies examined to date are associated with microsym- ations often dominate deep-sea communities depend- bionts, and in the case of 'bacteriosponges' the sym- ing on chernosynthesis, locally resulting in a high den- bionts may constitute more than one-third of the total sity of invertebrates. These oases of have a fauna1 tissue volume (Vacelet 1975, Reiswig 1981). However, biornass several orders of magnitude higher than that until very recently, associations with methanotrophic of the surrounding (Grassle 1986, Tunnicliffe bacteria were not described in the Porifera. 1991). The discrete participation of sponges in deep-sea Sponges had not previously been documented ecosystems directly depending on among the dense assemblages of invertebrates thriv- may be due to the absence of adaptations of these ing in effl.uents (Tunnicliffe 1991), sedentary organisms to life in such harsh, rapidly changing environments (Grassle 1986). However, it is also possible that sponges are more abundant in these assemblages than previously thought, given the poor knowledge of the distribution, biology and cytology of

0 Inter-Research 1996 Resale of full at-tidenot permitted Mar Ecol Proy Ser 145: 77-85, 1996

deep-sea sponges. Recently, associations between for direct transmission of the bacteria through genera- and methanotrophic bacteria occurring tions. near methane sources have been reported in the deep sea (Harrison et al. 1994, Vacelet et al. 1995) and in the deep , Irkutsh (Efremova et al. 1995). The MATERIALS AND METHODS association apparently results in an unusual abun- dance of sponges near the hydrocarbon sources. Sampling. Specimens (Fig 1) were collected from Here we describe one of these associations, which mud volcano fields in the Barbados Trench during the was first reported, in 1995 (Vacelet & Boury-Esnault 'Baresnaut' (1987) and 'Manon' (1992) cruises (Henry 1995) The , a carnivorous sponge without an et al. 1990, Le Plchon et al. 1990, Olu et al. in press). aquiferous system, is abundant around deep-sea mud Field observations and collections were made by volca.noes near the Barbados accretionary prism. It means of the submersible 'Nautile' (IFREMER)on the harbors 2, possibly 3, morphological types of bacteria. diatreme Atalante during the d.i.ves Manon MAO1 The tissue 6'" and diNvalues, the presence of a key (13" 49' 36" N, 57" 39' 58" W, 4943 m) and Baresnaut of methanotrophy in the sponge tissue, the PL 94/3 (13" 49' N, 57" 38' W, 4935 m), and on the habitus and distribution of the , and the ultra- diapir Mount Manon during the dive Manon MA06 structure of some symbionts all support the thesis that (13" 46' 74"N. 57" 32' 51" W, 4718 m). at least some of the symbionts are methanotrophic. Immediately after recovery, small samples of the Ultrastructural evidence is presented for intracellular specimens from the Manon cruise were either pre- phagocytic of the bacteria by the sponge and served for cytology or frozen. The remaining speci-

Fig. 1. Cladorhiza sp. Large bush of cladorhizid sponges, approximately 40 cm high, observed from the submersible 'Nautile' at 4943 m in the mud volcano Atalante in the Barbados Trench. The bushes were most abundant and larger around the periphery of the mud-volcano where the largest amounts of methane were detected (OIFREMER) Vacelet et al.: Bacteria-sponge symbios~s 79

mens, approximately 100 individuals, were preserved nized around a dense central axis of spicules. It con- in alcohol. The sponge is a new of Cladorhiza tained ovoid embryos 250 pm in maximum diameter. (Demospongiae, ) which will be de- The tissue contained 2, possibly 3, morphological types scribed in a forthcoming paper. Reference specimens of bacteria, distinctly different in size and ultrastruc- have bee.n deposited in the Museum National d'His- ture. Both common types were present in several dif- toire Naturelle in Paris (no. MNHN D JV 57). ferent extra- or intracellular locations throughout the Cytology. The specimens were fixed in 3% glu- sponge. The lateral appendages of the sponge, which taraldehyde in 0.4 M cacodylate buffer (pH 7.4) and were not examined by electron microscopy, appeared postfixed in 1% osmiun~tetroxide in the same buffer. under light microscopy to have the same organization They were dehydrated through an alcohol series and as the body. No gradient in the structure and distribu- embedded in Araldite. Thin sections were cut after tion of bacteria and bacteriocytes in the diverse zones local desilicification in 5 % hydrofluoric acid applied on of the sponge body was discernible. No difference was the free surface of the trimmed block (Borojevic & Levi observed between the samples from the 2 locations 1967). Semi-thin sections were stalned with toluidine (diatreme Atalante and Mount Manon). blue. Thln sections were contrasted with uranyl Extracellular symbionts were found at low density acetate and lead citrate, and observed under a Hitachi in the matrix of the sponge body among fib- Hu 600 or Zeiss EM 912 transmission electron micro- rils (Fig. 2A, B). Their distribution was patchy, al- scope. though large clusters of symbionts were occasionally Tissue analyses. Key of methylotrophic and seen. A rough estimate of the volume of the various chemoautotrophic sulfur-oxidizing bacteria were tissue components made from TEM micrographs indi- tested as follows. RUBISCO (EC 4.1.1.39) activity was cated that the extracellular bacteria never exceed assayed by the method of Wishnick & Lane (1971) as 12% of the total tissue volume in the areas examined. modified by Felbeck (1981).Methanol dehydrogenase When extracellular, the symbionts were intact with no was assayed by the method of Anthony & Zathman slgns of degradation, and division stages were ob- (1965).ATP sulfurylase (EC 2.7 7.4)and ATP reductase served (Fig. 2B). (EC 1.8.99.2) were determined spectrophotometrically The symbionts were also abundant intracellularly in as described in Felbeck (1981). the sponge (Fig. 2C. D). Symbionts were Tissue stable carbon and nitrogen isotope analyses observed in all types, including the sclerocytes were conducted on freeze-dried and acidified samples which secrete the siliceous spicules. The symbionts using standard methods with combustion techniques occurred either singly or loosely grouped in vacuoles by J. Boulegue and A. Mariotti (Universite P. et M. of diverse size (Fig. 2C, D),or crowded in large, round Curie, Paris, Laboratoire de Geochimie et Chimie vacuoles (Fig. 3). The different symbiont types were isotopique). The analyses were accomplished on a generally found in separate inclusions or vacuoles, Finningan Delta E mass spectrophotometer. Carbon is although they were occasionally seen together. Many reported relative to PDB (PeeDee Belernnite) and of the intracellular symbionts appeared to be in various nitrogen to atmospheric molecular nitrogen. stages in degradation, especially those crowded in the large vacuoles, suggesting lysosomal digestion by the host cell. Because of their d~stinctultrastructure, the RESULTS larger bacteria with stacked lamellae were easily rec- ognizable in various stages of degradation in these Cytology vacuoles (Fig. 3). The degrading bacteria had a more electron-opaque , more closely packed The sponge (Fig. 1) 1s 4 to 40 cm high and has th.e stacked lamellae and an irregular shape. In the most hydroid-like appearance typical of the , erect advanced stages, the bacteria were reduced to myelin- and branching with a cylindrical body 1 to 1.25 mm in like figures or simply the (Fig. 3A). Degrada- diameter from which arise numerous lateral branches tion of the smaller symbionts was more difficult to fol- 2 to 3 mm long and 0.12 to 0.25 mm in diameter low due to their lack of visually distinct ultrastructural Although it is devoid of an aquiferous system, as is features (such as stacked membranes and electron- usual in cladorhizids (Sars 1872, Lundbeck 1905), no lucent vacuoles), but was occasionally apparent part of the sponge tissue is distant from the external (Fig. 3A). The symbionts in a given vacuole of a bacte- medium due to the thinness of both body and lateral riocyte were usually of consistent appearance; how- branches. ever, vacuoles with symbionts in different stages of The tissue of the sponge body was found to be com- d~gestionwere often present in the same cell (Fig. 3). posed of various types of rounded or stellate cells, The symbionts were also observed within flattened whose ultrastructural preservation was irregular, orga- cells which formed a thin layer at the surface of 80 Mar Ecol Prog Ser 145: 77-85, 1996

Fig. 2. Extracellular and intracellular lnicrosymbionts in Cldd~rhj~dsp. tissues. (AITwo ultrastructural types of bacteria are present in the intercellular space: (1)Type I with stacked lamellae and (2)smaller rod-shaped cells. A third morphological type (3)was observed very rarely. Scale bar = 1.2 pm. (B) Division stage of an extracellular microsyrnblont (Type lmethanotroph). Scale bar = 0.29 pm. (C) Vacuoles containing a single type of microsyrnbiont (small rod-shaped bactena). Scale bar = 1.28 pm. (D) Vacuoles containing both types of microsymbionts, although bacteria with stacked lamellae are dominant. Scale bar = 1.28 pm embryos (Fig. 4). Only a single developmental stage of always contained singly within a vacuole. No obvi- the embryo was observed and it is not known whether ously degrading or dividing symbionts were observed these cells are embryonic cells or follicle cells from the in this location. Symbionts were absent from the large mother sponge surrounding the embryos. The first internal cells of the embryo, whi.ch contained numer- interpretation is more likely as the flattened-cell layer ous and inclusions. is located inside a dense collagen layer surrounding Two, possibly 3, ultrastructural types of symbiotic the embryo. In the embryo cells, the symbionts were bacteria with Gram-negative-type cell walls were Vacelet et al.: Bacteria-sponge symbiosis 81

Fig. 3. Intracellular degradation of microsymbionts of Cladorhiza sp. (A)A sponge cell containing vacuoles with Type 1 rnethan- otrophs in various stages of degradation. Small rod-shaped bacteria are less clearly recognizable (arrows). Note the large num- ber of electron-lucent granules in several bacteria, possibly poly-hydroxy-butarate, and the inclusion with myelin-like figures (*), presumably a lysosornal residual body. Scale bar = 1 pm. (B)Apparently synchronous degradation of Type I in 2 vacuoles of a sponge cell. Scale bar = 0.7 pm

present in the sponges (Fig. 5): (1) The most abun- Enzymes dant were coccoid cells, 1 to 1.7 pm in diameter (Fig. 5A, C). They contained stacks of flattened vesi- Significant methanol dehydrogenase activity was cles, approximately 20 nm in width, arranged in par- found in each of the 3 tissue samples analyzed (0.35, allel in the center of the cell. The cells also contained 0.48, and 0.56 international units g-' mix'). These levels small dense granules and a variable number of spher- are comparable to those found in the gill tissue of mus- ical or irregular vacuoles in their penpheral cyto- sels harboring methanotrophic bacteria (Cavanaugh plasm. Electron-lucent droplets, 0.1 to 0.15 pm in dia- 1993, Fisher 1993).This is strong evidence for the pres- meter, were often visible in intracellular symbionts, ence of abundant methanotrophic symbionts in this but rare in extracellular symbionts (Fig. 3A). This sponge (Nelson & Fisher 1995). Low activities of RuBP morphology is typical of Type I methanotrophic bac- carboxylase were detected in the samples (0.001inter- teria (Davies & Whittenbury 1970, Higgins et al. national units g-' min-'), which could be due to either 1981), and reminiscent of the methanotrophic sym- Type X methanotrophs, low activity in chemoautotrophic bionts seen in several species of deep-sea mytilids symbionts, or surface bacteria. Neither ATP sulfurylase (Cavanaugh 1993, Fisher 1993). (2) The second type nor APS reductase activities were detectable in any of were smaller, oval to rod-shaped cells, 0.25 to the 3 tissue samples (limits of detection for these assays 0.85 pm in diameter, 1.5 pm in maximum length, were between 0.1 and 0.2 international units g-' min-l). without intracellular membranes (Fig. 5B). This type often displayed a clear, microfibrillar central area with a dense core (Fig. 2A, C) (3) Cells approxl- Stable isotopes mately 1 pm in diameter, without stacked membranes and with a large, clear central area, were very rarely The tissue 6I3C values were very similar in the 3 observed in localised zones of the mesohyl (Fig. 2A). sponge samples analyzed (-48.4,-48.5, -48.8%). These Mar Ecol Prog Ser 145 77-85, 1996

sufficient symbionts are present to contribute signifi- cantly to the sponges' nutntion. Ultrastructural obser- vations indicate that the nutritional relationship between host and symbionts is based on A similar mechanism has been suggested for a variety of other chemoautotrophic and methanotrophic sym- b~oses(Fiala-Medioni 1988, Fiala-Medionl et al. 1989, Fiala-Medioni & Felbeck 1990, Fisher & Childress 1992, Nelson & Fisher 1995). As discussed below, the peculiarity here is that the symbionts are normally extracellular, with nutrient transfer between host and symbionts occurring through phagocytosis and intra- cellular digestion of bacterial cells within most of the sponge cell types. The distribution and habitus of the sponges also pro- vide evidence for nutrit~onalreliance of the sponge on methane. Deep-sea cladorhizids are normally sparsely distributed and occur in low densities as discrete indi- viduals, either anchored by rhizoids in muddy sedi- ment or attached to small debris. The population den- sity of representatives of the genera Cladorhiza and has been estimated from photographs Fig 4 M~crosymb~onts~n the embryo of Cladorhlza sp The em- and images to be to individuals ha ' In the bryo IS enveloped by a dense layer of collagen f~brlls(CF) The abyssal zone near Clarion and Cllpperton in the tropl- si~rfacecells contaln healthy rod-shaped bactena (arrows) and cal Pacific (T~lot1992) This contrasts with the abun- ~~peI methanotrophs (arroxvhead) occurring singly In men]- dance and size of Cladorhlza sp observed in the pre- brane-bound vacuoles The embryo Internal cells, xvith numer- sent study around the mud volcanoes The sponge ous yolk lncluslons do not contain symblonts Scale bar = 1 6 pnl occurred In dense clumps, up to 2 m In diameter and 40 cm high, of several hundred ~ndividuals(Fig 1) in values are consistent wlth incorporat~onof methane into the central depression, or eye, of the diatreme Ata- the sponge, as a methdne hydrate collected from the lante The central depress~onitself was f~lledwith same site was -58 1 * 0 2 " (Henry et a1 In press), and warm mud and was the site of the most active methane methane from the pore flu~don the volcanoes was -60 to expulsion (Le Pichon et a1 1990 Olu 1996, Olu et a1 in

-65' !r, (Martln et a1 in press) Tlssue S 'N values (+ 2 0, press) The sponges weie most abundant at the imme- 2 4, and 2 9" ) were relatively light and lower than what dlate periphery (0 to 100 m, 'leopard fac~es',Le Pichon has been reported for normal deep-sea fauna, but well et a1 1990) of the eye, mostly on the east side Smaller withln the range reported for other vent and seep fauna bushes (0 2 to 0 5 m in diameter) were abundant (Fisher et a1 1994, Van Dover & Fry 1994) between 100 m and 250 m from the edge of the eye ('yellow facies') No sponges lvere seen on the dia- treme Cyclope, In which the estimated methane flux

DISCUSSION (60 kg d '1is less than 1 %, of that of the d~atremeAta- lante (8000 kg d l) (Olu et a1 In press) The unusually The ultrastructure of the dominant morpholog~cal high dens~tyof very large aggregates of Cladorh~zasp type of symbiont and the level of methanol dehydroge- at the mud volcano s~te,with size and abundance of nase activity in the sponge tissues suggest that the the sponge bushes clearly correlated with the methane sponge symb~ontsare methanotrophic bactena The flow, also supports the thes~sthat the sponge derives hypothesis that the sponge denves some of its nutntion substantial benef~ts from the methane source Al- from its methanotrophlc symbionts 1s supported by the though the sponge, which 1s apparently devoid of the very negative SI3C values and the low F "N values normal cladorhizid rhizo~dsystem for growth on mud, which reflect significant input of methane and organlc may also benefit from the presence of a carbonate crust nitrogen of local orlgln The levels of methanol dehy- providing a substrate, it is unlikely that the avallab~l~ty drogenase In the sponge tlssue are comparable to of a hard substrate alone would result in such remark- those found in mussels which rely heavily on their able aggregations methanotrophlc symbionts for nutritional carbon The sponge morphology erect with branching pro- (Fisher & Chlldress 1992, F~sher1993) Indicating that cesses bearing a thick cover of hook-l~kemicrosclere Vacelet et al.: Bacteria-sponge s)mbiosis

Fig. 5. Structure of the microsymbionts of Cladorhiza sp. (A) Extracellular bacteria with stacked membranes (TypeI methan- otroph). Scale bar = 0.2 pm. (B) Small coccoid or rod-shaped bacteria in extracellular position. Scale bar = 0.2 pm. (C) Intracellu- lar Type I methanotroph, occurring singly in vacuoles. Scale bar = 0.25 pm spic.ules, suggests that the sponge may also feed on bacteria were relatively minor components of the small swlmming prey, as do other cladorhizids (Vacelet sponge tissue. It is also poss~blethat the low level of & Boury-Esnault 1995). This was supported by the RUBISCO activity detected was due to either Type X presence of debris from small , 0.5 to 1 mm methanotrophs or contaminating chemoautotrophic in length, trapped on the appendages of the sponges, bacteria associated with the surface of the sponge tis- and the number of 'associate' crustaceans and poly- sue samples. chaetes collected with the sponge (Bellan-Santini The symbionts are present in several different loca- 1990, Segonzac pers. comm.). Although originally con- tions, both extra- and intracellularly, in the sponge tis- sidered as associated fauna (Bellan-Santini 1990), the sue. Healthy, intact symbionts were found extracellu- crustaceans and were more probably prey larly in the sponge mesohyl. They were most often in in the process of capture or digestion. Although the the process of degradation when intracellular, with the relative importance of carnivory and methanotrophy notable exception of s.urface cells of the embryo. This is cannot be evaluated from these observations, it similar to what is observed in many other sponges sym- appears unlikely that such large aggregations of biotic with heterotrophic bacteria (Vacelet& Donadey carnivorous sponges could survive in the deep sea in 1977, Wilkinson 1987). In these other sponges, 1, 2, or the absence of added nutritional input. Feeding on a large number of morphological types of bacteria are free-living methanotrophic bacteria is unlikely, as present extracellularly within the sponge tissue, and cladorhizid sponges, devoid of an aquiferous system, are phagocytized and digested by specific sponge cell are not filter feeders (Vacelet & Boury-Esnault 1995). types. This is also observed in most phototrophic asso- Two morphological types of symbionts dominate the ciations between shallow-water sponges and unicellu- association, and the evidence indicates that at least lar . The peculiarity in this association one of these is methanotrophic. The current data are w~thmethanotrophs is the abundance of bacteria in the insufficient to draw conclusions with respect to the sec- process of degradation within all the sponge cell types. ond dominant morphological type of symbiont, the rod- The association is different from those of most other like bacteria. Dual symbioses with both chemoauto- invertebrates with methanotrophic or chemoauto- trophic sulfur bacteria and methanotrophic bacteria trophic sulfur-oxidizing symbionts, in which the sym- have been recognised in mytilid molluscan hosts bionts are generally housed within cells, where they (Fisher et al. 1993, Distel et al. 1995) and this is a possi- can both develop and occur in phagolysosomes (Fiala- bility in this association. However, the lack of Medioni & Metivier 1986, deBurgh et al. 1989, Fiala- detectable ATP sulfurylase or APS 1-eductase activity, Medioni & Felbeck 1990). However, similar complex coupled with the very low levels of RUBISCO activity, situations in which healthy extracellular chemoauto- suggest that if this is the case then the sulfur-oxidizing trophic bacteria are phagocytized and digested by epi- Mar Ecol Prog Ser 145: 77-85, 1996

dermal or gill cells are known in gutless oligochaetes Cary SC, Giovannoni SJ (1993) Transovanal Inheritance of (Giere & Langheld 1987) and in thyasirid molluscs endosymbiotic bacteria in clams ~nhabiting dt'c>p-sea (Southward 1986). hydrothermal vents and cold seeps. Proc Natl Acdd Scl USA 90:5695-5699 The presence of intact symbionts in cells at the sur- Cavanaugh CM (1992) Methanotroph- symbioses face of embryos suggests a mechanism for direct trans- in the marine environment: ultrastructural, biochvmical mission of the symbionts between generations. This is and molecular studies. In: Murrell JC. Kelly DP (edsj again reminiscent of littoral 'bacteriosponges', where Microbial growth on C1 compounds. Intercept Scientific, Andover, p 315-328 bacteria are transmitted between generations in Childress JJ, Fisher CR, Brooks JM, Kennicut I1MC, Bidigare (Levi & Levi 1976) or in follicle cells shed with R, Anderson AE (1986) A methanotrophic marine mollus- the oocytes (Gallissian & Vacelet 1976). A similar can (, ) symbiosis: mussels fueled by gas. mechanism of direct symbiont transmission has been Scicncc 233:1306-1308 suggested for clams with chemoautotrophic symbionts Davies SL, Whittenbury R (1970) Fine structure of methane and other hydrocarbon utilizing bacteria J Gen Microbial (Endow & Ohta 1990, Cary & Giovannoni 1993). 61.227-232 Two other apparently similar associations between deBurgh ME, Juniper SK. Singla CL (1989) Bacterial symb~o- sponges and methanotrophic bacteria have been re- sis in Northeast Paclf~cVestimentifera: a TEM study. Mar ported in preliminary papers: a sp. Biol 101 :97-105 Distel DL. Lee HKW, Cavanaugh CM (1995) Intracellular encrusting on vestimentiferan tube worms from hydro- coexistence of methano- and Lhioautotrophic bacteria in a carbon seep communities in the Gulf of Mexico, 600 m hydrothermal vent mussel. Proc Natl Acad Sci USA 92. deep (Harrison et al. 1994), and a fresh-water sponge, 9598-9602 Baikalospongia intermedia, from thermal vents of the Efremova SM, Fialkov VA, Kouzln VS (1995) Methanotrophic deep lake Baikal (Efremova et al. 1995). The fact that symbiotic bacteria are found in deepwater sponges. The Second Vereshchagin Baikal Conference. Irkutsk, Russia. such symbioses with methanotrophic bacteria exist in Octobrr 5-10, 1995. Abstracts, p 62-63 sponges widely differing in habitat, morphology, Endow K,Ohta S (1990) Occurrence of bacteria in the primary and feeding strategy (the marine Hyme- oocytes of vesicomyid clam Calyptogena magnifica. Mar desmia and fresh-water Baikalospongia being sponges Ecol Prog Ser 64:309-311 Felbeck H (1981) Chemoautotrophic potential of the with a 'normal' aquiferous system and filter-feeding hydrothermal vent , Jones habit, whereas Cladorhiza is a stalked, carnivorous (Vestimentifera). Science 213:336-338 sponge without an aquiferous system) could mean that Fiala-Medioni A (1988) Synthese sur les adaptations struc- methanotrophic associations are frequent in sponges turales Liees h la nutrition des mollusques bivalves des from methane-rich environments, although they have sources hydrothermales profondes Oceanol Acta 8 173-179 not been previously recognized. Fiald-Medioni A, Felbeck H (1990)Autotrophic processes in in- nutrition: bacterial symbiosis in bivalve molluscs. In: Kmne RKH, Kinne-Saffran E, Beyenbach KW (eds) Com- Acknowledgements. We are especially grateful to Prof. X. Le parative . Vol5. Karger, Basel, p 49-69 Pichon for providing the opportunity to dive and collect the Fiala-Medioni A, Felbeck H, Childress JJ. Fisher CR. Vetter sponges, as well as to the scientlflc crew of the 'Baresnaut' RD (1989) Lysosomic resorption of bacterial symbionts in and 'Manon' crulses (1987 and 1992). We also thank the 'Nau- deep-sea bivalves. In: Nardon P, Gianlnnazi-Pearson V, tlle' and 'Atalante' crews for the11 efficient cooperation. The Grenler AM, Maryulis L, Smith DC (eds)Endocytobiology. tissue stable carbon and n~trogenanalyses were conducted by Vol4. Villeurbanne p 335-338 J. Boulegue and A. Manotti. We thank C. Bezac and M. Fiala-~MedioniA. Metivier C (1986) Ultrastructure of the gill of Streams for technical assistance. This work was supported by the hydrothermal vent bivalve Calyptogena rnagn~fica. INSU/CNRS, URA 117, URA 41. OTAN CRG 9310552 and with considerations on its nutrition. Mar Biol 90:215-222 NSF EAR9158113. 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This article was submitted to the editor Manuscript first received: July 16, 1996 Revised version accepted: Octobet 21, 1996